This invention addresses the pressing need in biomedical science for imaging and tissue processing methods which can quickly image multiple components in thick tissues and whole organs with high molecular specificity. In order to develop methods which achieve this goal, the choice of an appropriate imaging technology is critical. Important criteria include spatial resolution, sensitivity, depth penetration, molecular specificity, data throughput and compatibility with the many biochemical methods such as immunohistochemistry or FISH analysis. Many methods to image 3D thick tissues have been developed, but most of these techniques do not have subcellular resolution or the necessary molecular sensitivity. For instance, while high resolution MRI is capable of imaging whole animals, its resolution is limited to 10-100 μm and is not compatible with common fluorescent markers. In comparison, optical imaging methods in general provide the highest resolution and specificity. Optical coherence tomography has a few-micron level resolution and a penetration depth of a few millimeters, however, optical coherence tomography does not provide reliable subcellular level imaging today. Optical projection tomography is more compatible with molecular imaging and can study fluorescent and non fluorescently stained tissues up to approximately 15 mm. However it also does not possess sufficient resolution to resolve details of individual cells and has difficulty in imaging tissues containing opaque materials such as bone or cartilage. Sheet plane illumination techniques, such as selective plane illumination microscopy (SPIM) has demonstrated the ability to provide detailed images of over a millimeter of relatively transparent samples such as embryos but, due to the residual scattering that exists even in optically cleared samples, SPIM and similar techniques have limitations with opaque samples and with samples which have an extent over several millimeters or which possess weak fluorescent signals. Further, since it is not generally possible to immunostain whole organs due to the limited diffusivity of antibodies and even of small molecules into intact tissues, sheet plane techniques are of limited use without first sectioning the tissue to allow the penetration of the appropriate labels. This restricts their use in answering many biological questions which require either IHC or FISH analysis to reveal relevant biomarkers.
Among 3D tissue optical imaging techniques, two-photon microscopy (TPM) is particularly promising. TPM is a fluorescent optical microscopy technique. It features sub-micron resolution, low photo-toxicity, excellent penetration depth, and 3D sectioning capability. The excellent depth penetration of TPM in tissues is due to lower scattering and absorption of the infrared excitation wavelength employed and the lack of the need for a detection pinhole allowing greater signal collection efficiency than in confocal microscopy. In addition, like all fluorescence based techniques, it provides high molecular specificity in mapping gene and protein expression profiles, and has clearly demonstrated its utility for visualizing gene activity in vivo with GFP over the past decade.
While, two-photon microscopy (TPM) can image in highly scattering media, it is still limited to approximately less than a millimeter penetration into opaque samples. To overcome the depth penetration limitation of two-photon microscopy in studying thicker specimens, preferred embodiments of the inventions use an automated microtome integrated into a high speed TPM system. By alternating and overlapping optical sectioning with mechanical sectioning, it is possible to rapidly image samples with arbitrary thickness. Once the upper portion of the sample is imaged, the uppermost portion of the tissue sample can be removed by the microtome. A critical problem with mechanically sectioned tissues, however, is the difficulty in comparative analysis due to stretching, compressing and/or rotation of tissue structures caused by mechanical sectioning. Prior methods have used fiducial markers formed by drilling holes or otherwise altering the tissue to aid in alignment.
While direct intravital tissue labeling, transgenic animals, native tissue autofluorescence, and SHG contrast provide powerful means to visualize the complex 3D biochemical environment within a tissue, there are still a large number of biochemical states and signatures which are only possible to examine by other means, such as immunohistochemistry (IHC) staining. Unfortunately it is very difficult or impossible to reliably IHC stain whole mount tissues greater than approximately 100 microns in depth. This is due to the large size of antibodies used in IHC staining and their subsequent slow diffusion and steric hindrance within the tissue.